Retrobiosynthesis of D-glucaric acid in a metabolically engineered strain of Escherichia coli

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2010.Cataloged from PDF version of thesis.Includes bibliographical references (p. 173-181).Synthetic biology is an evolving field that emphasizes synthesis more than observation which has been and is the scientific method for traditional biology. With powerful synthetic tools, synthetic biologists seek to reproduce natural behaviors (and eventually to create artificial life) from unnatural molecules or try to construct unnatural systems from interchangeable parts. Accompanied with this recent movement, metabolic engineers started to utilize these interchangeable parts (enzymes in this case) to create novel pathways. In addition, engineering biological parts including enzymes, promoters, and protein-protein interaction domains has led to productivity improvement. However, understanding behaviors of a synthetic pathway in an engineered chassis is still a daunting task, requiring global optimization. As the first step to understand pathway design rules and behaviors of synthetic pathways, a synthetic pathway for the production of D-glucaric acid has been designed and constructed in E. coli. To this end, three disparate enzymes were recruited from three different organisms, and the system perturbed by this introduction of heterologous genes was analyzed. Limiting flux through the pathway is the second recombinant step, catalyzed by myo-inositol oxygenase (MIOX), whose activity is strongly influenced by the concentration of the myo-inositol substrate. To increase the effective concentration of myo-inositol, synthetic scaffold devices were built from protein-protein interaction domains to co-recruit all three pathway enzymes in a designable complex.(cont.) This colocalization led to enhancement of MIOX activity with concomitant productivity improvement, achieving 2.7 g/L of D-glucaric acid production from 10 g/L of D-glucose input. Secondly, retrobiosynthetic approach, a product-targeted design of biological pathways, has been proposed as an alternative strategy to exploit the diversity of enzymecatalyzed reactions. The first step in a glucaric acid pathway designed retrosynthetically involves oxidation of the C-6 hydroxyl group on glucose, but no glucose oxidase in nature has been found to act on this hydroxyl group on glucose. To create glucose 6- oxidase, a computational design and experimental selection was combined with the help of DNA synthesis technology. To this end, the sequence space of candidate mutations was computationally searched, the selected sequences were combinatorially assembled, and the created library was experimentally screened and characterized. Furthermore, the structure-activity relationship of the newly created glucose oxidases was elucidated, and the kinetic model mechanism for these mutants was proposed and analyzed. Collectively, parts, devices, and chassis engineering were applied to a synthetic pathway for the production of D-glucaric acid, and this synthetic biology approach was proven to be effective for new pathway design and improvement.by Tae Seok Moon.Ph.D

Developing a novel microbial host and synthetic biology tools for valorizing waste polyethylene terephthalate and lignin-derived compounds

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BIOMECHANICAL ANALYSIS A SEQENCE OF ANGULAR VELOCITY AND COORDINATED MUSCLES ACTIVITY DURING BASEBALL HITTING

The purpose of this study was to analyse a sequence of rotations and coordinated muscles activities of upper body. Using kinematic and EMG data from 3 recreational university baseball players participating in this study, we computed the angular velocity of trunk, pelvis, bat and trunk-pelvis rotation angle and PMT of upper body muscles. Trunkpelvis rotation angle was 22 ° before the bat-ball contact. The pelvis, trunk, and bat showed a sequence of angular velocity beginning with the hip, followed by the trunk, and end tip of the bat. Additionally, PMT of upper body muscles generated right pectoralis major(1.03 sec.), right external oblique(1.11 sec.), left thoracloumbar fasci(1.12 sec.), left external oblique(1.13 sec.), right latissimus dorsi(1.15 sec.), left latissimus dorsi(1.16 sec.), right thoracloumbar fascia(1.16 sec.), left pectoralis major(1.25 sec.), on at a time during baseball hitting motion. PMT of upper body muscles were related to the shifting and rotating of body segment and this action can be considered the coordinated muscle activities of upper body

De novo design of heat-repressible RNA thermosensors in E-coli

RNA-based temperature sensing is common in bacteria that live in fluctuating environments. Most naturally-occurring RNA thermosensors are heat-inducible, have long sequences, and function by sequestering the ribosome binding site in a hairpin structure at lower temperatures. Here, we demonstrate the de novo design of short, heat-repressible RNA thermosensors. These thermosensors contain a cleavage site for RNase E, an enzyme native to Escherichia coli and many other organisms, in the 5′ untranslated region of the target gene. At low temperatures, the cleavage site is sequestered in a stem–loop, and gene expression is unobstructed. At high temperatures, the stem–loop unfolds, allowing for mRNA degradation and turning off expression. We demonstrated that these thermosensors respond specifically to temperature and provided experimental support for the central role of RNase E in the mechanism. We also demonstrated the modularity of these RNA thermosensors by constructing a three-input composite circuit that utilizes transcriptional, post-transcriptional, and post-translational regulation. A thorough analysis of the 24 thermosensors allowed for the development of design guidelines for systematic construction of similar thermosensors in future applications. These short, modular RNA thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications

COMPARISON OF PROPRIOCEPTION PERCEPTION TEST BETWEEN GOLFER AND NON-GOLFER USING TILTING PLATFORM

The purpose of this study was to test proprioception perception and compare between collegiate golfers and non-golfers using tilting platform. Sixteen male and fourteen female golfers and fifteen male and thirteen non-golfers were participated. All participants were performed perception test on the tilting platform. Frequency analysis and independent t-test were performed using SPSS 24.0. Alpha set at .05. Most participants were perceived from 1° to 2° of slopes and perceived left-right (target direction) slope than forward-backward slope. Repeated practice such as walking on the uneven ground or standing on sloped ground might help to improve proprioception perception. Further research using a tilting platform will be to develop the training program

Genetically stable CRISPR-based kill switches for engineered microbes

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EFFECTS OF GENDER AND FOOT POSITION ON ACCELERATION PATTERN OF KNEE AND HIP JOINT DURING DEEP SQUAT

The purpose of this study was to investigate the effect of gender and foot position on the acceleration patterns of the knee and hip joints during deep squat. Twenty-two male and 10 female collegiate students participated in this study. All the participants performed a deep squat two times in neutral foot position (NFP), with the foot rotated externally by 15° (ERFP). A wireless triaxial accelerometer was attached on the right-side knee and hip joints of each participant. Acceleration data generated in the anterior-posterior (AP), medio-lateral (ML), and superior-inferior (SI) directions during deep squat were collected through the attached acceleration sensor (2000Hz). Statistical analysis was performed using SPSS 24.0, and mixed analysis of variance (p \u3c 0.05) was used to identify the interaction and main effects of gender and foot positions. The acceleration patterns of the knee joint during deep squat according to gender indicated differences between the AP and ML directions. The acceleration motion of the hip joint under the ERFP condition indicated a difference in the SI direction

Optical spectroscopic investigation on the coupling of electronic and magnetic structure in multiferroic hexagonal RMnO3 (R = Gd, Tb, Dy, and Ho) thin films

We investigated the effects of temperature and magnetic field on the electronic structure of hexagonal RMnO3 (R = Gd, Tb, Dy, and Ho) thin films using optical spectroscopy. As the magnetic ordering of the system was disturbed, a systematic change in the electronic structure was commonly identified in this series. The optical absorption peak near 1.7 eV showed an unexpectedly large shift of more than 150 meV from 300 K to 15 K, accompanied by an anomaly of the shift at the Neel temperature. The magnetic field dependent measurement clearly revealed a sizable shift of the corresponding peak when a high magnetic field was applied. Our findings indicated strong coupling between the magnetic ordering and the electronic structure in the multiferroic hexagonal RMnO3 compounds.Comment: 16 pages including 4 figure

Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli

The field of metabolic engineering has the potential to produce a wide variety of chemicals in both an inexpensive and ecologically-friendly manner. Heterologous expression of novel combinations of enzymes promises to provide new or improved synthetic routes towards a substantially increased diversity of small molecules. Recently, we constructed a synthetic pathway to produce d-glucaric acid, a molecule that has been deemed a “top-value added chemical” from biomass, starting from glucose. Limiting flux through the pathway is the second recombinant step, catalyzed by myo-inositol oxygenase (MIOX), whose activity is strongly influenced by the concentration of the myo-inositol substrate. To synthetically increase the effective concentration of myo-inositol, polypeptide scaffolds were built from protein–protein interaction domains to co-localize all three pathway enzymes in a designable complex as previously described (Dueber et al., 2009). Glucaric acid titer was found to be strongly affected by the number of scaffold interaction domains targeting upstream Ino1 enzymes, whereas the effect of increased numbers of MIOX-targeted domains was much less significant. We determined that the scaffolds directly increased the specific MIOX activity and that glucaric acid titers were strongly correlated with MIOX activity. Overall, we observed an approximately 5-fold improvement in product titers over the non-scaffolded control, and a 50% improvement over the previously reported highest titers. These results further validate the utility of these synthetic scaffolds as a tool for metabolic engineering.United States. Office of Naval Research (Young Investigator Program, Grant No. N000140510656)Synthetic Biology Engineering Research CenterNational Science Foundation (U.S.) (Grant No. EEC-0540879)National Science Foundation (U.S.) (Grant No. CBET-0756801

Genetically stable CRISPR-based kill switches for engineered microbes

Microbial biocontainment is an essential goal for engineering safe, next-generation living therapeutics. However, the genetic stability of biocontainment circuits, including kill switches, is a challenge that must be addressed. Kill switches are among the most difficult circuits to maintain due to the strong selection pressure they impart, leading to high potential for evolution of escape mutant populations. Here we engineer two CRISPR-based kill switches in the probiotic Escherichia coli Nissle 1917, a single-input chemical-responsive switch and a 2-input chemical- and temperature-responsive switch. We employ parallel strategies to address kill switch stability, including functional redundancy within the circuit, modulation of the SOS response, antibiotic-independent plasmid maintenance, and provision of intra-niche competition by a closely related strain. We demonstrate that strains harboring either kill switch can be selectively and efficiently killed inside the murine gut, while strains harboring the 2-input switch are additionally killed upon excretion. Leveraging redundant strategies, we demonstrate robust biocontainment of our kill switch strains and provide a template for future kill switch development